Circulating small extracellular vesicles in Alzheimer’s disease: a case–control study of neuro-inflammation and synaptic dysfunction

Background Alzheimer’s disease (AD) is a neurodegenerative disease characterized by Aβ plaques and neurofibrillary tangles. Chronic inflammation and synaptic dysfunction lead to disease progression and cognitive decline. Small extracellular vesicles (sEVs) are implicated in AD progression by facilitating the spread of pathological proteins and inflammatory cytokines. This study investigates synaptic dysfunction and neuroinflammation protein markers in plasma-derived sEVs (PsEVs), their association with Amyloid-β and tau pathologies, and their correlation with AD progression. Methods A total of 90 [AD = 35, mild cognitive impairment (MCI) = 25, and healthy age-matched controls (AMC) = 30] participants were recruited. PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy. Using nanoparticle tracking analysis, the size and concentration of PsEVs were determined. Antibody-based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies. Synaptic dysfunction and neuroinflammation were evaluated with synaptophysin, TNF-α, IL-1β, and GFAP antibodies. AD-specific markers, amyloid-β (1–42), and p-Tau were examined within PsEVs using Western blot and ELISA. Results Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p < 0.0001). Amyloid-β (1–42) expression within PsEVs is significantly elevated in MCI and AD compared to AMC. We could also differentiate between the amyloid-β (1–42) expression in AD and MCI. Similarly, PsEVs-derived p-Tau exhibited elevated expression in MCI compared with AMC, which is further increased in AD. Synaptophysin exhibited downregulated expression in PsEVs from MCI to AD (p = 0.047) compared to AMC, whereas IL-1β, TNF-α, and GFAP showed increased expression in MCI and AD compared to AMC. The correlation between the neuropsychological tests and PsEVs-derived proteins (which included markers for synaptic integrity, neuroinflammation, and disease pathology) was also performed in our study. The increased number of PsEVs correlates with disease pathological markers, synaptic dysfunction, and neuroinflammation. Conclusions Elevated PsEVs, upregulated amyloid-β (1–42), and p-Tau expression show high diagnostic accuracy in AD. The downregulated synaptophysin expression and upregulated neuroinflammatory markers in AD and MCI patients suggest potential synaptic degeneration and neuroinflammation. These findings support the potential of PsEV-associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-024-03475-z.

Methods A total of 90 [AD = 35, mild cognitive impairment (MCI) = 25, and healthy age-matched controls (AMC) = 30] participants were recruited.PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy.Using nanoparticle tracking analysis, the size and concentration of PsEVs were determined.Antibody-based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies.Synaptic dysfunction and neuroinflammation were evaluated with synaptophysin, TNF-α, IL-1β, and GFAP antibodies.AD-specific markers, amyloid-β , and p-Tau were examined within PsEVs using Western blot and ELISA.

Results
Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p < 0.0001).Amyloid-β (1-42) expression within PsEVs is significantly elevated in MCI and AD compared to AMC.We could also differentiate between the amyloid-β (1-42) expression in AD and MCI.Similarly, PsEVs-derived p-Tau exhibited elevated expression in MCI compared with AMC, which is further increased in AD.Synaptophysin exhibited downregulated expression in PsEVs from MCI to AD (p = 0.047) compared to AMC, whereas IL-1β, TNF-α, and GFAP showed increased expression in MCI and AD compared to AMC.The correlation between the neuropsychological tests and PsEVs-derived proteins (which included markers for synaptic integrity, neuroinflammation, and disease pathology) was also performed in our study.The increased number of PsEVs correlates with disease pathological markers, synaptic dysfunction, and neuroinflammation.*Correspondence: Saroj Kumar saroj.kumar@associated.ltu.se;sarojgupta.k@gmail.comFull list of author information is available at the end of the article Conclusions Elevated PsEVs, upregulated amyloid-β , and p-Tau expression show high diagnostic accuracy in AD.The downregulated synaptophysin expression and upregulated neuroinflammatory markers in AD and MCI patients suggest potential synaptic degeneration and neuroinflammation.These findings support the potential of PsEV-associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression.Keywords Alzheimer's disease, Mild cognitive impairment, Small extracellular vesicles, Synaptic dysfunction, Neuroinflammation
In recent years, small extracellular vesicles (sEVs) or exosomes have been acknowledged as crucial mediators of communication and signaling within the body, contributing significantly to the transmission of cellular cargo in various health and disease states.They also play a notable role in disseminating protein aggregates associated with neurodegenerative diseases [32].sEVs are bi-layered membrane vesicles that have a heterogeneous group of (< 200 nm in diameter) that are found in different human body fluids, including blood, urine, saliva, and ascites, and that are actively released by all cell types [33][34][35].For their functions in various physiological and pathological circumstances, sEVs are the most extensively researched type of EV [36][37][38].sEVs exchange information between cells by transferring bioactive components (nucleic acids and proteins) [39].As the sEVs' composition bears the molecular signature of the secreting cell and bears an intrinsic property of transversing the blood-brain barrier (BBB) in both directions [40,41], they are a target of constant research in neurodegenerative disease.Furthermore, sEVs released by neuronal cells are crucial in transmitting signals to other nerve cells, influencing central nervous system (CNS) development, synaptic activity regulation, and nerve injury regeneration.Moreover, sEVs exhibit a dual function in neurodegenerative processes, as sEVs not only play an essential role in clearing misfolded proteins, thereby exerting detoxifying effects and providing neuroprotection [42].On the other hand, they also have the potential to participate in the propagation and aggregation of misfolded proteins, particularly implicated in the pathological spread of Tau aggregates as indicated by both in vitro and in vivo studies [43].As a protective mechanism, astrocytes (most abundant glial cells) accumulate at the locations where Aβ peptides are deposited, internalizing and breaking down aggregated peptides [44].However, severe endosomal-lysosomal abnormalities arise in astrocytes when a significantly large amount of Aβ accumulates within astrocytes for a prolonged period without degradation [45,46].Astrocytes then release engulfed amyloid-β (1-42) protofibrils through exosomes, leading to severe neurotoxicity to neighboring neurons [44].Additionally, it has been found that the release of amyloid-β by microglia in association with large extracellular vesicles (Aβ-lEVs) damages synaptic plasticity and modifies the architecture of the dendritic spine [47].Thus, sEVs can be a compelling subject for the investigation to understand AD's inflammation and synaptic dysfunction [48][49][50][51][52].
In this study, we reported that protein levels are associated with AD pathology, neuroinflammation, and synaptic dysfunction in plasma-derived small extracellular vesicles (PsEVs).Our objective was to understand the pathophysiological process, neuroinflammation, synaptic dysfunction, and Aβ pathology through sEVs.Our study revealed a significant correlation between the concentration of cargo proteins derived from PsEVs and clinical diagnosis concerning ACE-III and MMSE scores.Furthermore, the levels of these studied proteins within PsEVs could differentiate between patients with MCI and AD.Thus, our study sheds light on the potential of PsEVs in understanding AD dynamics and offers insights into the underlying mechanisms of disease progression.

Subject recruitment
A total of n = 35 AD patients and n = 25 subjects with MCI were recruited from the Memory Clinic, Department of Geriatrics, All India Institute of Medical Sciences, New Delhi, India.Additionally, n = 30 healthy AMC (volunteers) were recruited.The inclusion criteria were as follows: a clinical diagnosis of MCI and AD patients using ACE-III and MMSE tests.The exclusion criteria encompass medical conditions such as cancer, autoimmune disorders, liver disease, hematological disorders, or stroke, as well as psychiatric conditions, substance abuse, or any impediment to participation.Controls were healthy, age-matched adults without neurological symptoms.AMC was 60-71, MCI was 65-79, and AD was 70-80 years of age range (Table 1).
Neuropsychological scores, viz., ACE-III and MMSE, were recorded before subject selection.

Study ethical approval
The institutional ethics committee of All India Institute of Medical Sciences, New Delhi, India, granted the study ethical permission.The study has been granted the ethical approval number IECPG-670/25.08.2022.Following the acquisition of the written informed consent, all participants were enrolled.

Sample collection
One milliliter of blood was drawn from each participant using venipuncture, and blood collection vials were kept on ice during collection.The blood was centrifuged at 1700 g for 20 min at 4 °C to remove the cells, and the straw-colored plasma was collected.It was further clarified by centrifuging for 30 mi at 4 °C at 10,000 g.Finally, cleared plasma was stored at − 80 °C until further use.The samples were used for the downstream experiment after being thawed on ice and centrifuged at 10,000 g.

Isolation of PsEVs
The PsEVs were extracted by chemical-based precipitation from the plasma samples of AD patients, MCI patients, and AMC, as discussed previously [53,54].In brief, 180 μL of plasma sample was used and filtered with 0.22 μm filter (SFNY25R, Axiva), followed by overnight incubation with the chemical precipitant (14% polyethylene glycol 6000) (807,491, Sigma).The samples underwent an hour-long, 13,000 g centrifugation at 4 °C the next day.Before being resuspended in 200 μL of 1X PBS (ML116-500ML, HiMedia), the pellet was first cleaned twice with 1X PBS.Before downstream experiments, the sEVs-enriched fraction was further filtered through a 100-kDa filter (UFC5100, Millipore).

Nanoparticle tracking analysis (NTA)
5000-fold dilution in 1X-PBS buffer was used for the NTA of PsEVs.In the ZetaView Twin system (Particle Metrix, Germany) sample chamber, 1 mL of diluted PsEVs sample was introduced.The following parameters were used throughout three cycles of scanning 11 cell locations each, and 60 frames per position were collected (video setting: high, focus: autofocus, shutter: 150, 488 nm internal laser, camera sensitivity: 80, cell temperature: 25 °C.CMOS cameras were used for recording, and the built-in ZetaView Software 8.05.12 (Particle Metrix, Germany) was used to analyze: 10 nm as minimum particle size, 1000 nm as maximum particle size, and 30 minimum particle brightness.

Transmission electron microscopy for morphological characterization
Transmission electron microscopy was employed to investigate PsEVs' ultrastructural morphology.The resultant PsEVs pellet was diluted with PBS using 0.1 M phosphate buffer (pH 7.4).A carbon-coated copper grid of 300 mesh (01843, Ted Pella) was used to adsorb the separated PsEVs at room temperature for 30 min.After blot-drying, the adsorbed grids were dyed.For 10 s, 2% aqueous uranyl acetate solution (81,405, SRL Chem) as negative staining.After blotting the grids, they were inspected using a Talos S transmission electron microscope (ThermoScientific, USA).

Enzyme-linked Immunosorbent Assay (ELISA)
According to the previous protocol, ELISA was carried out.[53].PsEV samples were subjected to freeze-thaw cycles; next, PsEVs were ultrasonicated for two minutes, with a 30-s on-and-off cycle, at an amplitude of 25.Following this, they underwent a 10-min centrifugation at 10,000 g, at 4 °C, and the obtained supernatant was used.

Data and statistical analysis
The mean age values, ACE-III score, and MMSE score were ascertained using descriptive statistical analysis Table 1.GraphPad Prism 8.0 was used for statistical data analysis, including NTA concentration, Western blotting densitometric analysis, and ELISA.Unpaired student t-test and ANOVA were used for group analysis, and statistical significance was determined.p < 0.05 was used to assess significance.The Image J software (NIH, USA) was used for the densitometry analysis.The receiver operating characteristic (ROC) curve was used to analyze the efficiency of distinguishing the case from controls.Correlation analysis was conducted between the concentration of PsEVs and the levels of ELISA proteins, including amyloid-β (1-42), p-Tau, IL-1β, TNF-α, GFAP, and synaptophysin, and additionally between the PsEVs-derived levels of amyloid-β (1-42) β1-42, p-Tau, IL-1β, TNF-α, GFAP, and synaptophysin with ACE-III and MMSE values.ROC curve is a probability curve utilized to assess the accuracy of a test.The test's ability to distinguish between groups is indicated by the area under the curve (AUC), which acts as a quantitative measure of separability.An outstanding test typically exhibits an AUC close to 1, signifying a high level of separability.Conversely, a subpar test tends to have an AUC closer to 0, indicating a poor ability to distinguish between the two classes.
For proinflammatory markers, IL-1β and TNF-α levels showed a significant increase among the three groups (p < 0.0001 for IL-1β and TNF-α) (Fig. 3I, M).When comparing AD to MCI and AMC, the GFAP concentration in PsEVs was significantly higher (p < 0.0001) (Fig. 3Q).Similar trends were observed with Western blot analysis (Fig. 2, Additional File 1: Fig. S6, S9).Their elevated levels suggest prominent neuroinflammatory conditions contributing to potential neuronal damage.The elevated levels of these neuroinflammatory markers could be due to the activation of astrocytes and microglia and the subsequent increase in the secretion of PsEVs containing proinflammatory proteins, which suggests prominent neuroinflammatory conditions that may contribute to neuronal damage [57].While synaptophysin concentration in PsEVs was downregulated in AD and MCI compared to AMC (p < 0.0001) in ELISA (Fig. 3U), it shows synaptic dysfunction.We also checked synaptophysin levels in PsEVs in Western blotting, finding it was downregulated in AD compared to MCI and AMC (p = 0.0045, 0.0142), indicating synaptic degeneration in AD (Fig. 2, Additional File 1: Fig. S5).In MCI, synaptophysin levels did not significantly differ from AMC (Fig. 2F).This aligns with synaptic loss in AD, reflected in lower neuropsychological test scores indicating more pronounced cognitive impairment compared to MCI and AMC.

Discussion
In this study, we aimed to investigate the capacity of PsEVs to mirror pathological processes linked to AD and MCI.sEVs are extensively documented in the propagation of pathological processes associated with neurodegenerative and metabolic disorders [60].The increased secretion of sEVs, coupled with the transmission of disease-related pathologies through sEVs-associated cargo, makes sEVs a viable candidate for understanding the physiological state of their originating cells, which is reflected in sEVs cargo [61].To isolate the PsEVs, we employed a combined approach involving chemical precipitation followed by ultrafiltration, which effectively eliminates co-precipitants and minute protein contaminants such as albumin and LDL.We employed the neuronal protein L1CAM as a marker to ascertain the neuronal origin, although there is a debate surrounding its specificity for neuronal origin [62].Nevertheless, in our study, the L1CAM marker is used to check for protein markers and not to confirm L1CAM affinitybased isolation.A two-step filtration procedure was used to accompany the sEV isolation method in our study to ensure high purity.Spherical lipid bi-layered vesicles within the typical size range of small extracellular vesicles (30-150 nm) were observed across AD, MCI, and AMC subjects (Fig. 1A-C).NTA was employed to study the size distribution of sEVs in AD, MCI, and AMC.We observed that the isolated PsEVs come within the size range of < 200 nm, and there was a notable increase in the number of particles in diseased conditions compared to the control group.(Fig. 1D-G).
Validation using sEVs-specific markers (CD63, CD81, and TSG101) demonstrated a noteworthy upregulation in MCI and AD, indicating PsEVs numbers are increased in disease conditions (Fig. 2A-D).Levels of sEV-specific markers in AD and MCI are elevated because PsEV numbers are increased in the disease condition.As documented by various studies in MCI and AD, there is an increase in cross-talk between different pathophysiological processes, which leads to an increase in sEVs number and sEVs specific marker as a cellular response to heightened cellular stress aggravating neuronal damage and synaptic dysfunction [33,63,64].Neuroinflammation, a characteristic feature of AD and MCI, may lead to the release of sEVs with inflammatory markers.Synaptic dysfunction, evidenced by synaptic degeneration, could contribute to the increased sEV-specific markers, reflecting vesicle release in response to altered synaptic activity [9,65].Additionally, cells undergoing stress might activate compensatory mechanisms, and the elevated sEV-specific markers could signify communication for potential repair or damage mitigation.Therefore, the increase in sEV-specific markers may be linked to the progression of neurodegenerative processes, indicating ongoing pathological changes in the brain as the disease progresses.Additionally, the elevated expression of L1CAM, a neuron-associated marker, in MCI and AD further strengthens the association between PsEVs and Table 3 Correlation analysis of PsEVs-derived amyloid-β (1-42), p-Tau, IL-1β, TNF-α, GFAP, and synaptophysin with neuropsychological scores neurodegenerative processes (Fig. 2E).Furthermore, our observations extend beyond AD and MCI, showing increased concentrations of sEVs in other health conditions where higher levels of these vesicles correlate with elevated levels of disease markers [53][54][55].The results of our research provide valuable insight into the characterization, validation, and functional implications of plasma-derived small extracellular vesicles (PsEVs) in the context of AD and MCI.Our comprehensive analysis encompassed morphological, biochemical, and functional aspects, shedding light on the potential role of PsEVs as biomarkers and contributors to neurodegenerative processes.
For this purpose, we performed the ELISA of amyloid-β  in PsEVs, where we observed higher protein concentrations of amyloid-β (1-42) in MCI.At the same time, in AD, the concentration also significantly increased (Fig. 3A).In a similar study by A. Manolopoulos et al. [66], they studied levels of Aβ42, total Tau, and pro-brain-derived neurotrophic factor (BDNF) in both plasma neuron-derived extracellular vesicles (NDEVs) and plasma.The study reported a lack of correlation between the plasma and NDEVs, substantiating concerns about levels of the Aβ42 and total Tau measured in plasma originating from non-CNS sources.Multiple studies support the involvement of extracellular vesicles (EVs) in AD pathogenesis, where Aβ and Tau are released in association with EVs, influencing neuronal cell death and trans-synaptic spreading of the disease [7,15,54,67].A progressive elevation in PsEV levels of p-Tau was observed in MCI, reaching a significantly higher AD concentration (Fig. 3E).Previous research has revealed that p-tau alone effectively differentiates Frontotemporal Dementia (FTD) from AD with high specificity [68,69].In our study, the alone analysis of p-Tau and amyloid-β (1-42) proved effective in distinguishing patients with MCI from AMC (Table 2).Consequently, studies have reported that the elevation of p-Tau suggests the future likelihood of AD development [70].This dual elevation in amyloid-β (1-42) and p-Tau levels highlights their potential utility as concurrent biomarkers associated with MCI and AD diagnosis, as indicated by our ROC analysis.Therefore, the investigation into PsEV content revealed significant alterations in key markers associated with AD pathology, viz., amyloid-β (1-42) and p-Tau, which are a well-established marker of AD and exhibit an elevated level in PsEVs from AD and MCI patients compared to AMC in our study.
Synaptic dysfunction is considered a core feature of AD.It is suggested to precede other pathophysiological events of AD rather than neurodegeneration, which manifests during the later stages of the disease [71].Synaptic dysfunction interacts with other core pathophysiology events of AD, such as the amyloid-β cascade, tau pathology, and neuroinflammation, eventually progressing to irreversible neurodegeneration and atrophy [72,73].In this context, the synchronized exchange of proteins involved in these pathological processes between the CNS and neuronal-derived sEVs highlights the potential of sEVs as reliable carriers of pathophysiological cascade occurring at the pathological site [74].In Fig. 3U, we observed downregulated synaptophysin levels, a synaptic vesicle marker, in AD PsEVs compared to MCI and AMC.This suggests synaptic degeneration, which has also been discussed in several studies [59,63,64].Synaptic damage induced by amyloid-β deposition triggers a response from the glia to eliminate impaired synapses.As amyloid-β accumulates, the severity of synaptic dysfunction intensifies, leading to tau hyperphosphorylation and the formation of tau tangles.Our study's findings contradict J. Utz et al. (2021), which showed increased synaptophysin levels in microvesicles isolated from cerebrospinal fluid (CSF) in AD [28].This discrepancy could be due to different biofluid sources, cellular origins, or clearance mechanisms for synaptophysin in these compartments.Our study also differs from Utz J et al. (2021) as we have studied PsEVs compared to microvesicles; both differ in biogenesis, structure, and functions.Moreover, our study aligns with existing studies that reported lower synaptophysin levels in plasma neuronal-derived EVs.Goetzl et al. [75] investigated the synaptic protein levels in neuronal-derived exosomes in plasma (NDEs) of patients with FTD and AD, where the authors found significantly lower levels of synaptopodin, neurogranin, synaptophysin, and synaptotagmin-2 in both conditions compared to controls.Furthermore, our results also align with the overall synaptic loss seen in AD patient's brains, where lower levels of synaptophysin in the hippocampus have been reported to correlate with cognitive decline in AD [59].Our study found that no significant difference in synaptophysin levels between MCI and AMC was observed, indicating that synapse dysfunction is more pronounced due to neuronal loss in the advanced disease stage, and its indication is reflected in PsEVs.Since the PsEVs pool also contains neuronal-derived EVs, we interpolate that the reduction in synaptic proteins in brain tissue is reflected in our results.
IL-1β, a potent immunomodulating cytokine, has previously been identified as a trigger for various inflammatory mediators in astrocytes and neurons [76].Consistent evidence from post-mortem AD brain studies indicates the prevalent overexpression of IL-1β, with immunohistochemical analyses revealing its localization to microglia around plaques [77].Moreover, pro-inflammatory markers (IL-1β and TNF-α) were significantly higher in PsEVs from AD and MCI subjects, as evidenced by ELISA and Western blot findings in our study (Fig. 3I and M).Table 3 summarizes the correlation between PsEVs and neuroinflammatory markers.IL-1β plays a direct role in the pathophysiological changes associated with AD owing to its specific expression in the vicinity of plaques, and this localization suggests IL-1β as a mediator in the formation of plaques and tangles, thereby contributing to AD pathology [65].TNF-α, another pro-inflammatory cytokine primarily secreted by activated macrophages and microglia, is recognized for its dual role in promoting cell survival and death in the central nervous system [78,79].
The cytoskeletal GFAP is found in astrocytic cells [80].Increased plasma GFAP levels could result from "reactive astrogliosis, " another term for aberrant astrocytic function brought on by damage to neurons [81].According to research on animal and cell models, reactive astrocytes encircle and penetrate amyloid-β plaques, contributing to the amyloid-β pathological process [82,83].Research has demonstrated a correlation between amyloid-β burden, cognitive decline, and plasma GFAP [83].PsEVs of GFAP were elevated in AD [27] and MCI (Fig. 3Q).It is well known that sEVs play a pivotal role in the progression of disease pathologies in neurodegenerative and metabolic diseases [33,84].The high levels of neuro-inflammatory markers (GFAP, TNF-α, and IL-1β) in PsEVs from MCI and AD subjects suggest a potential role of PsEVs in neuroinflammation.This activation of astrocytes and microglia precedes increased secretion of pro-inflammatory PsEVs and may contribute to neuronal damage and progressive cognitive impairment.Diseased conditions involve an increased secretion of sEVs and the cargo they carry, including pathological hallmark proteins or immunomodulatory cytokines [33].
Correlation analyses unveiled positive associations between PsEVs concentration and the protein levels of amyloid-β (1-42), p-Tau, IL-1β, TNF-α, GFAP, and synaptophysin (Fig. 4).Furthermore, our study also analyzed the correlation between cognitive examination scores (ACE-III and MMSE) and PsEV-associated protein levels (Fig. 5).The negative correlations observed imply that lower cognitive scores align with elevated levels of amyloid-β (1-42), p-Tau, IL-1β, and TNF-α in PsEVs Table 3.This implies a strong connection between PsEV biomarkers and cognitive decline, reinforcing that PsEVs could serve as valuable diagnostic and prognostic tools.These findings underscore the potential of PsEVs as reliable disease progression and pathology indicators.The robust correlations further support the hypothesis that PsEVs may actively participate in disseminating neurodegenerative signals.
Our study extensively studied the multiple pathophysiological processes associated with AD by checking the protein levels involved in these processes within PsEVs, including amyloid-β (1-42), p-Tau, neuroinflammatory markers (IL-1β, TNF-α, GFAP), and synaptic protein levels.This comprehensive approach enhances diagnostic accuracy by considering the synergistic effects of these processes, providing valuable insights into disease progression from MCI to AD.We have also performed a systematic comparison with MCI, which was lacking in previous studies.We observed a significant correlation between these investigated protein levels within PsEVs and neuropsychological tests, thus filling a research gap addressing the clinical relevance of these dysregulated pathophysiological processes.The observed downregulated synaptophysin levels in AD PsEVs compared to MCI and control subjects shed light on the combined role of neuroinflammation and proteinopathy in the cognitive decline observed as the disease progresses.This finding suggests that PsEVs may reflect synaptic degeneration, opening avenues for further exploration into the role of PsEVs in synaptic damage and dysfunction in neurodegenerative diseases.

Conclusions
Our study provides a multifaceted examination of PsEVs, offering compelling evidence of their potential as biomarkers and functional contributors in AD.We have comprehensively discussed the synergistic role of synaptic dysfunction and neuroinflammation and their association with amyloid-β and tau pathologies within the PsEVs in AD progression.The pathophysiological conditions in the MCI and AD brain are reflected in PsEVs, as observed by the increased concentration of PsEVs containing disease-associated markers and markers for synaptic dysfunction and neuroinflammation.Therefore, the PsEVs can be exploited to understand the pathophysiological process involved in the progression and severity of MCI and AD.
Fig. 1D-F, the size distribution and concentration of PsEVs were observed in the size range of 30-200 nm in diameter by NTA, which is within the sEVs' size range.The mean concentration of PsEVs in AMC, MCI, and AD

Fig. 1
Fig. 1 Isolation and analysis of PsEVs.The isolated PsEV morphology characterize by transmission electron microscopy from age-matched healthy controls (AMC) (A), mild-cognitive impairment (MCI) patients (B), and Alzheimer's disease (AD) (C).The size distribution of PsEVs subpopulation (nm) versus the concentration (particle/ml) in AMC (D), individuals with MCI (E), and AD (F).Comparison of the sEVs concentration of AD, MCI, and AMC patients (G).Receiver operating characteristic (ROC) curve of PsEVs concentration in AMC v/s AD (H), AMC v/s MCI (I), and MCI v/s AD (J) (scale bar 100 nm)

Table 1
Demographic details of samples